User manual LEICA CONFOCAL APPLICATION LETTER BROCHURE 02-2007

[. . . ] All FRAP experiments discussed below were performed with a Leica TCS SP5 confocal microscope. Instead of fluorescent proteins (FPs) the HaloTag Interchangeable Labelling Technology, which is based on non-fluorescent HaloTag protein, was used. Signals were detected by incubation with various fluorescent ligands, which are able to covalently bind to the HaloTag protein. In FRAP experiments fluorescent molecules are bleached by a laser pulse in a selected region of a specimen and the subsequent increase of fluorescence is measured in the same region. If fluorescent molecules diffuse through the specimen, kinetic parameters of a protein can be determined by the increase of fluorescence. [. . . ] FRAP experiments and data analysis were done with the Leica FRAP application wizard. The experimental settings of the confocal microscope are displayed in table 1. Results For the FRAP experiments, cytoplasmic regions of stained cells were chosen and selectively bleached. The loss of fluorescence caused by image acquisition was corrected in the data and normalized with respect to the prebleach intensity. Transmitted light images were recorded in parallel to demonstrate the cell shape (Fig. Cells stained with the HaloTag-diAcFAM ligand showed a decrease in fluorescence by 35% of the original intensity after the bleaching pulse. This experiment represents other experiments with HaloTag-diAcFAM stained cells. In all experiments, the half-time recovery of 1. 14 +/- 0. 38 seconds was observed. Cells stained with HaloTag TMR ligand were inves-tigated under the same conditions recovered with a half-time of 1. 24 +/- 0. 15 seconds, a slightly slower kinetic (Fig. For image acquisition before and after bleaching, the DPSS 561nm laser was used. To achieve more effective bleaching, the 488 nm Argon laser was additionally applied only for A B C D E F Figure 1: A­C, -Tubulin-HaloTag fusion protein stained with HaloTag-diAcFAM ligand. (A, D) fluorescence, (B, E) transmitted light, (C, F) Overlay of fluorescence- and transmitted light image. A B 1. 1 1 0. 9 0. 8 0. 7 0. 6 0. 5 0. 4 0. 3 0. 1 0 0 5 10 15 20 25 30 35 [s] (1) (3) (2) Figure 2: A, FRAP experiment with -Tubulin-HaloTag fusion protein stained with HaloTag-diAcFAM-Ligand. B, 1) last prebleach, 2) bleach pulse with 488 nm argon-laser line, 3) last postbleach. Confocal Application Letter 3 FRAP Leica Microsystems Heidelberg GmbH · Am Friedensplatz 3 · D-68165 Mannheim, Germany · Tel. +49 (0) 6 21-70 28-0 · E-Mail: clsm. support@leica-microsystems. com LEICA and the Leica Logo are registered trademarks of Leica IR GmbH. Here the initial fluorescence intensity decreased more than 80% and after 30 seconds the fluorescence reached 85 % of the initial fluorescence (Fig. Non-transfected cells were as control of the specificity of the staining with both ligands. With the excitation wavelengths mentioned above the cells did not show any detectable fluorescence (data not shown) either for diAcFAM or for TMR. Discussion The use of new specific fluorescence ligands[5-7] with different spectral characteristics allows for more flexibility to detect non-fluorescent proteins. Timeconsuming and cumbersome sub-cloning work can be avoided with the described methods. However, potential toxicity and specificity need always to be taken into account when applying these technologies. [. . . ] (1976): Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers. (2001): From fixed to FRAP: measuring protein mobility and activity in living cells. (1998): Fluorescent labeling of recombinant proteins in living cells with FlAsH. (2003): A general method for the covalent labeling of fusion proteins with small molecules in vivo. [. . . ]